Methods

Nine native T. graeca and 9 alien T. horsfieldii, all 1 year of age, were selected from a breeding farm in Lebanon (33.5°N, 35.5°E, 0 m altitude). Tortoises of each species were placed in a separate room (7.5 m²), with daily monitoring of temperature and humidity. The respective average temperature and relative humidity were 17°C and 64% for the cold months (December–February), and 22.5°C and 75% for the warm months (March–April). Light and temperature were controlled through automatically timed neon lamps, providing 13 light hours a day (0500–1800 hours) throughout the experimental period, and infrared heat lamps that were kept on for 24 hours a day throughout the cold months (December–February). The room's cement floors were covered by wood shavings that were changed twice during the 6-month period of the experiment. In addition, some rocks provided a shelter for the tortoises.

Blood (1 mL) was sampled from the heart of the tortoises with a 27-gauge (0.4 mm × 13 mm) tuberculin needle that was introduced through soft tissues in the axillary region at the base of the forelimbs (Gandal 1958; Stephens and Creekmore 1983; Taylor and Jacobson 1981). Anesthesia was not used. Individual Wright-Giemsa–stained blood smears were prepared by using the method of Kolmer et al. (1959). A contact time of 1 minute between the stain and the blood cells was allowed before adding a buffer solution of monobasic potassium phosphate and dibasic sodium phosphate. The mixture was allowed to remain over the smeared cells for 4 minutes, before flooding with tap water for 30 seconds. A Giemsa stain was then poured and kept on each slide for 2 minutes, then washed with tap water and dried at room temperature. Differential cell counts of 10 randomly chosen fields of each individual smear were done at a magnification of ×400.

The remaining blood was injected into lithium heparin–containing microcapillary tubes, which were placed in an IEC MB centrifuge (DAMON/IEC Division, MA, USA) and spun at 12,700 × g for 5 minutes for separation of plasma from blood cells. The capillary tube was cut to remove the packed blood cells, and the remaining part of the capillary, which contained the plasma was sealed from both ends by crytoseal cement and preserved at −20°C. The preserved plasma was later used for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to determine the banding pattern of serum polypeptides and their color intensities. In addition, a portion of the plasma was used to study the antiproliferation and cytotoxicity effect on human colon cancer cells (HCT-116).

The SDS-PAGE was used to compare the banding pattern of the plasma polypeptides of the 2 tortoise species and the intensity of each banded polypeptide (Barbour et al. 1989). A Mini PROTEAN II electrophoresis cell (Bio-Rad Laboratory, Richmond, CA, USA) was assembled according to the procedure described by the Bio-Rad Manual (Bio-Rad Laboratory, Richmond, CA, USA). A 12% separating gel was prepared and allowed to polymerize for 45 minutes, and the stacking gel polymerization for 60 minutes. Four individual plasma samples from each of T. horsfieldii and T. graeca were diluted 1:40 with the sample buffer, without the addition of SDS; whereas, the low-range marker of 14.4–97.4 kDa was diluted 1:40 with the stock sample buffer supplemented with SDS. A volume of 20 μL of each plasma sample and 5 μL of the marker were loaded on individual lanes of the stacking gel. Electrophoresis was performed at 60 mA for 45 minutes. The gel was stained with 0.1% Coomassie blue for 30 minutes with continuous shaking. The background was destained for 2 hours. Gels with banded polypeptides were photographed by using the Gel Doc system (Bio-Rad Laboratories, Inc, Hercules, CA, USA). This system allowed the determination of the density of the developed color in each band in pixel units.

A preliminary pilot study screened plasma of 5 randomly chosen individual T. graeca collected in winter and another 6 collected in spring for comparison of their antitumor bioactivities in human cancer cell lines. HCT-116 human colon cancer cells were grown in RPMI 1640 at 37°C in a 5% CO₂ incubator. The HCT-116 cell culture medium was supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum. The seeding of the HCT-116 cells in 6-well plates was at the level of 2.0 × 10⁴ cells in 100-μL wells, incubated for 24 hours at 37°C before treatment with tortoise plasma. Triplicates of each tortoise plasma, diluted to 5%, were each applied in 5-μL volumes on the HCT-116 cell culture. The percentage HCT-116 cell proliferation and percentage viability were determined by using the respective Cell Titer 96 nonradioactive cell proliferation assay and the Cytotox 96 nonradioactive cytotoxicity assay, according to the instructions of the manufacturer (Promega Corp, Madison, WI, USA) (Moravec 1994). The proliferation assay is an MTT (Methyl Thiazolyl Tetrazolium)-based method that measures the ability of metabolically active cells to convert tetrazolium salt into a formazan product, and its absorbance is recorded at 570 nm. The Cytotox 96 assay quantitatively measures the lactate dehydrogenase (LDH), a stable cytosolic enzyme that is released upon cell lysis. Released LDH in culture supernatants is measured with a coupled enzymatic assay, which results in the conversion of a tetrazolium salt into a red formazan product, the absorbance of which is recorded at 490 nm. Control wells were included in each of the 2 assays by depriving the HCT-116 from contact with tortoise plasma, thus establishing 2 reference points of 100% proliferation and 100% viability, respectively. A second investigation compared the effect of 3 different concentrations of tortoise plasma (5%, 10%, and 15%) of 7 randomly chosen individual T. graeca on the average percentage viability of HCT-116 human colon cancer cells. Each concentration of each individual plasma sample was run in triplicate. Controls were included, and the viability assay was performed as described above.

Computerized statistical analysis was performed by using the MSTAT software (MSTATC, 1991, Michigan State University, MI, USA). The experimental design was of a 2-factor completely randomized structure. One-way analysis of variance of the different measured parameters was performed. Means were then separated by Duncan's multiple range test (α = 0.05).

Results and Discussion

Results showed no significant difference (p > 0.05) between T. horsfieldii and T. graeca in the percentage of red blood cells (RBC), thrombocytes, heterophils, basophils, lymphocytes, monocytes, and eosinophils in either winter or spring (Tables 1 and 2). Others (Taylor and Jacobson 1981; Lawrence and Hawkey 1986) have shown a drop in the RBC and leukocyte count at the beginning of hibernation (fall season), a period that is not included in this study. Future work should include the 4 seasons of the year and other years in the life of tortoises. The absence of a significant difference between the hematologic parameters obtained in this experiment could be related to the slight difference in the average temperature and relative humidity between winter (December–February) and spring (March–May). In addition, the average temperature in this study for the winter season was relatively high compared with other conditions mentioned in other studies, namely that of Lawrence and Hawkey (1986) who conducted their experiment in Britain where winter temperatures are significantly lower.

Table 1. Average percentage of red blood cells (RBC) and thrombocytes in 2 different tortoise species in their second year of life during winter and spring.^a

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Table 2. Average percentage of white blood cells in 2 different tortoise species during winter and spring (see Table 1 for definition of abbreviations).^a

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The respective intensities in pixel units of each of the 9 banded plasma polypeptides in T. horsfieldii vs. T. graeca are presented in Table 3. There were some statistical differences in the intensities of certain polypeptide bands when compared with the 2 species. The presence of 9 banded polypeptides in each of 4 plasma individual serum samples of T. horsfieldii (lanes 1–4) is shown in Fig. 1; however, each of the 4 individual plasma samples of T. graeca showed only 7 banded polypeptides (Fig.1, lanes 1–4). Plasma polypeptide profiles can be used as support for the differentiation of animal species (Lykakis 1971; Dangerfield et al. 1976). This new finding of the presence of 2 additional polypeptide bands in T. horsfieldii, namely, the 26.1 kDa and 68.3 kDa bands, enables us to add this character as differentiating this species from T. graeca. The existence of these 2 polypeptide bands in T. horsfieldii needs further investigation for its genetic basis, and the biological function of these 2 polypeptides.

Table 3. Average^a intensity of different plasma polypeptide bands in 2 different tortoise species (see Table 1 for definition of abbreviations).

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Results of the study on the bioactivity of T. graeca plasma on HCT-116 human colon cancer cells showed that a concentration of 5% plasma collected in the winter induced significant differences (p < 0.05) in percentage proliferation and percentage viability of the targeted human colon cancer cells (Table 4). The range of cancer cell percentage proliferation induced by the 6 individuals of T. graeca widened to 46.37%–99.96%. A significant difference in impact of plasma on cancer cell line viability was also present in the winter; however, the range was narrower, at 94.84%–99.27%. In contrast, the plasma collected during spring did not show significant variation among the individuals regarding both the percentage proliferation and the percentage viability (p > 0.05) (Table 4). Moreover, the average impact of all individual plasma on percentage proliferation and percentage viability of cancer cells did not differ significantly between the winter and spring (p > 0.05).

Table 4. Impact of the 5% concentration of T. graeca (TG) tortoise plasma collected in winter and spring on HCT-116 colon cancer cells (see Table 1 for definition of abbreviations).

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These preliminary data need further investigation to uncover the reason behind the significant differences among individuals of T. graeca regarding the impact of the plasma collected in the winter season on the percentage proliferation and percentage viability of human colon cancer cells. As the concentration of the plasma collected from 7 individual T. graeca increased from 5% to 15%, the percentage viability of the cancer cells was significantly reduced, from 76.95% to 53.23% (p < 0.05) (Fig. 2). This anticancer effect was clearly a plasma concentration-dependent relationship. Future investigations will target the anticancer-amplification mechanisms in tortoise plasma that could result in more significant reduction of percentage viability of HCT-116 human colon cancer cells and will also focus on identification of the anticancer factor present in certain individuals of T. graeca.

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